Aminopeptidases are found in various microorganisms and animal cells, and generally metallo-proteases requiring a metal ion such as calcium and zinc for their activities. Said enzymes are known to be capable of maintaining their activities of diverse nature, depending on their origin of derivation, even in a temperature range of 50.degree. to 70.degree. C.
Aminopeptidases have their industrial uses in the determination of protein primary structures, and in the pharmaceutical drug development etc., and, in particular, they are useful for the preparation of a recombinant protein having a same amino acid sequence as its wild-type protein by eliminating N-terminal methionine from the recombinant protein, which is accompanied by methionine in its N-terminal(N-methionyl recombinant protein), produced in a microorganism by using a recombinant DNA technique.
As the presence of an N-terminal methionine on recombinant human proteins may cause an unwanted immune reaction when administered to human beings, it would be desirable to remove the N-terminal methionine, thus producing the mature eucaryotic proteins whose primary amino acid sequences are identical to that of human origin.
G. Roncari and H. Zuber disclosed in Method in Enzymology, 19, 544(1970) three aminopeptidases derived from Bacillus stearothermophilus, having slightly different properties from each other; and Takayuri Uwajima et al. reported in Agr. Biol. Chem., 37(12), 2727(1973) the physicochemical properties of an aminopeptidase requiring a calcium ion for maintaining its activity, which was derived from Streptomyces peptidofaciens KY 2389.
In addition, various aminopeptidases have been obtained from, e.g., Bacillus subtilis (Fred W. Wagner et al., Arch. Biochem. Biophys., 197(1), 63-72(1979)), Streptomyces rimosus (Lj. Vitale et al., Appl. Microbiol. Biotechnol., 23, 449-455(1986)), Streptomyces griseus (K. D. Vosbeck et al., J. Biol. Chem., 238, 6029-6034(1973)), etc.
A. Spungin and S. Blumberg disclosed in Eur. J. Biochem., 183, 471-477(1989) an aminopeptidase having a molecular weight of about 33 kd as determined by SDS-PAGE and N-terminal amino acid sequence of Ala-Pro-Asp-Ile-Pro-Leu (SEQ ID NO: 1), which was separated and purified by a process which comprises heat-treating a culture of Streptomyces griseus separated from soil and subjecting the heat-treated culture to Bio-Gel P-4 gel filtration chromatography and DEAE Sepharose ion exchange chromatography.
Recently, an enzyme called pronase, separated from Streptomyces griseus K-1 strain, has become commercially available. However, since the enzyme contains various proteinases as a mixture, it is not efficient to remove only the N-terminal methionine residue.
On the other hand, there have been many attempts made to prepare a wild-type protein by removing the N-terminal methionine residue from the recombinant protein produced in a microorganism. Exemplary methods thereof may include a process for preparing wild-type human growth hormone by expressing a fusion protein in which the N-terminal of human growth hormone is fused with the C-terminal of another protein, and then severing the fusion protein by using a specific protease(PCT International Publication No. WO 89/12678; EP Patent Publication Nos. 20290 and 321940); a process for preparing a wild-type recombinant protein by expressing a recombinant protein having a secretion signal peptide in a microorganism cell so that the secretion signal peptide containing the N-terminal methionine residue is removed when the protein is secreted out of the cell and then recovering the wild-type recombinant protein from the medium (EP Laid-open Publication No. 0088632 A2; U.S. Pat. No. 4,755,465; Japanese Patent Laid-open Publication No. 01273591; EP Patent Publication No. 306673; and Korean Patent Laid-open Publication No. 94-579). However, the above methods produce a low yield, have the difficulty of isolating the desired wild-type recombinant protein from the fusion protein if the two proteins have similar physical properties, and may require complicated and laborious procedures for transforming a host cell with a newly prepared expression vector and determining the optimum fermentation conditions.
It has been suggested that a wild-type recombinant protein may be prepared without suffering from such defects by using a specific aminopeptidase which is capable of specifically removing a methionine residue present in its N-terminal of N-methionyl recombinant protein(PCT International Publication Nos. WO 86/04609 and WO 86/204527 A1). For example, in the case of methionyl human growth hormone whose N-terminal amino acid sequence is Met-Phe-Pro-Thr-Ile (SEQ ID NO: 2), the N-terminal methionine may be removed selectively by using an aminopeptidase which can recognize the X-pro(wherein X means any amino acid residue) site at the N-terminal, thereby terminating its severing reaction before the X residue.
Hitherto, a limited number of aminopeptidases such as the one separated and purified from a culture of Vibrio proteolyticus(Bio-Technology General Corp., WO 86/01229) and the one extracted and purified from a porcine kidney (Takeda, WO 86/204527 A1) have been reportedly used for the preparation of a wild-type recombinant protein by removing its N-terminal methionine residue. However, there still exists a demand for the development of aminopeptidases which meet various substrate specificity and high enzyme activity requirements.